This project seeks to understand how newly synthesized secretory and membrane proteins are made, matured, sorted, and metabolized in cells. This class of proteins is essential to all intercellular and intracellular communication, and their precise locations and abundances are tightly regulated to maintain normal cellular and organismal physiology. Indeed, the majority of current drug targets affect secreted and membrane proteins, underscoring their central role in human biology. Our goals are to develop a molecular level understanding of the pathways of secretory and membrane protein biosynthesis and metabolism. Not only are we interested in these normal cellular events, but also in discovering the ways they are perturbed in certain disease states. Using the biosynthesis of mammalian Prion protein (PrP) as a model system, we have discovered that the most important and tightly regulated step in its initial segregatin to the endoplasmic reticulum (ER) is the interaction between its signal sequence and the protein translocon. This step was found to be critically dependent on a four protein complex of previously unknown function (termed the TRAP complex), in the absence of which PrP does not enter the ER. Our finding that not all signal sequences require TRAP suggests that different substrates are recognized differently by the translocon, an idea further supported by recent studies analyzing crosslinking between signal sequences and translocon components. More significantly, we have now shown that alterations in the nature of this signal-translocon interaction have substantial consequences for protein localization and function. In the case of PrP, the cellular burden of potentially cytotoxic forms can be reduced (or enhanced) to change the susceptibility of cells to otherwise harmful insults. In the case of other proteins (Calreticulin and p58-IPK), we find that signal-translocon interactions are critical in allowing these proteins to exist in two compartments (the ER lumen and the cytosol), where they could potentially serve independent functions. Thus, advances during the past few years are beginning to illuminate a novel site of potential cellular regulation, the entry of secretory and membrane protein substrates into the mammalian secretory pathway, that impacts both normal physiology and disease progression. Most recently, these insights have been applied to uncover the ways in which protein entry into the ER is modulated productively by the cell under conditions of stress. This analysis has led to the discovery of a new degradation pathway we have termed pre-emptive quality control (or pQC). The mechanisms that facilitate pQC and the degradative machinery are currently being studied in biochemical systems that recapitulate these events in vitro. In addition to mechanistic functional studies, we are engaged in collaborative studies to gain insight into the structural features of the mammalian ER translocon. Cryo-electron microscopy approaches are being combined with biochemical reconstitution to determine the structures of translocon complexes containing different components or arrested in different functional states. And finally, parallel collaborative studies are using in vivo systems to understand the physiologic importance of regulating translocation of specific proteins into the mammalian ER. In one project, we are examining the role of cytosolic calreticulin generated by translocation regulation in mice models. In another project, we are investigating the role that protein translocation of PrP plays in the various diseases associated with this protein.